A standard field of use of light microscopy for examining biological preparations is luminescence microscopy. Here, luminescence is understood, as is generally usual, as a generic term for phosphorescence and fluorescence, thus covers both processes. As predominantly fluorescence is used in microscopy, in this description the term fluorescence is often used, but is meant, insofar as technically meaningful, to serve only as an example of processes that generally utilize luminescence.
In luminescence microscopy, particular luminescence dyes (so-called phosphors or fluorophores) are used for the specific tagging of samples, e.g. of cell parts. Samples which luminesce per se, thus without added tagging substance, can also be surveyed. The sample is excited to luminescence with radiation (using so-called excitation radiation) and the excited luminescence light recorded with suitable detectors. For this, a dichroic beam splitter is usually provided in the light microscope in combination with block filters which split the luminescence radiation from the excitation radiation and enable a separate observation. Through this procedure, the representation of individual, differently coloured cell parts in the light microscope is possible. Of course, several parts of a preparation can also be simultaneously coloured with different dyes attaching specifically to different structures of the preparation. This method is called multiple luminescence.
For resolution in standard light microscopy, the wavelength of the radiation used is a determining variable, as according to Ernst Abbe within the framework of an imaging the wavelength used (together with the numerical aperture of the imaging lens system) predetermines the diffraction limit and thus the resolution.
Different approaches have recently been developed for resolutions beyond the diffraction limit. These microscopy methods are characterized by the fact that they provide the user with a higher lateral and/or axial optical resolution compared with the standard microscope. In this description a microscope is described as being of high-resolution if it reaches a resolution beyond the optical diffraction limit. Diffraction-limited microscopes, on the other hand, are called standard microscopes. They realize known optical wide-field microscopy or laser scanning microscopy.
Within the framework of luminescence microscopy, different high-resolution methods attempt to ensure that as far as possible only a volume which is smaller than a minimum volume predetermined by the diffraction limit luminesces. If the degree of volume reduction, e.g. due to the optical parameters of the irradiation, is known, it is known that acquired fluorescence radiation, irrespective of its diffraction-limited broadening during detection, comes from a volume reduced to below the diffraction limit, and a high-resolution image can thus be produced.
This method is used for example in DE 102006009833 A1 which uses so-called reversible saturable optical fluorescence transition (RESOLFT). This microscopy method makes use of a tagging substance which can be repeatedly transformed with the help of a switching beam from a first state in which the tagging substance can be excited to fluorescence into a second state in which no fluorescence can be produced. The named published document describes the state in which the tagging substance can be excited to fluorescence as “fluorescing state A”. Naturally, in this first state fluorescence occurs only if the sample is illuminated by excitation radiation, i.e. the sample per se does not fluoresce, but can be excited to do so. In this description, in order to make a clear distinction reference is made to a state that can be excited to fluorescence.
FIG. 1 of the named DE 102006009833 A1 shows in three part-figures a)-c) the three essential part-steps of the microscopy concept described there. The sample is first irradiated with switching radiation, with the result that the sample reaches the state A that can be excited to fluorescence. The illumination is carried out in wide-field (cf. FIG. 1a of the named published document). The sample is then illuminated with a standing-wave field which ensures that only area parts of the sample remain in the fluorescence-capable state A (cf. FIG. 1b of the named published document). Through the standing-wave field which is produced by an interference pattern, this concept means that the sample parts remaining in the fluorescence-capable state A are smaller than the diffraction limit of the optical imaging actually allows. The first two steps thus represent a sample preparation which prepares the sample such that only volume parts of the sample are in a fluorescence-capable state A, wherein, due to the standing-wave field, these parts are in themselves smaller than the diffraction-limited resolution of the optical imaging actually allows. In a last step the thus-prepared sample is then excited to fluorescence. Due to the prior sample preparation, only the parts of the sample which have remained in state A can then emit fluorescence radiation. DE 102006009833 A1 describes the excitation of the prepared sample for the emission of fluorescence radiation as “reading out the fluorescence”. For this reading out, i.e. for the excitation of the fluorescence, according to DE 102006009833 A1 the known TIRF concept can also be used in which the fluorescence of the preparation is excited via an evanescent field by irradiating the excitation radiation below the total reflection angle at the boundary between cover glass and sample. This principle known to a person skilled in the art is described for example in the publication by Thompson NL et al., “Measuring Surface Dynamics of Biomolecules by Total Internal Reflexion Fluorescence with Photobleaching Recovery or Correlation Spectroscopy”, Biophys J., 33, No. 3, 1981, pp. 435-454.